Table-1: Genes That Were Down Modulated in Pooled Samples of EB

Table-1: Genes that were down modulated in Pooled samples of EB

BG02-EB derived at day 13a

Gene / BG02-ES / Day 13-BG02- EB / PES / PEB
ES cell markers
Galanin / 33 / 4 / 5 / 4
POU5F1 / 28 / 3 / 23 / 3

GTCM-1

/ 14 / 12 / 15 / 9

GDF3

/ 13 / 2 /

4

/ -
GJA1 / 10 / 4 /

6

/ 2

Nanog

/ 6 / -b /

8

/ -

Cell cycle regulation

Lin-28 / 28 / 13 /

13

/ 10

SEMA6A

/ 25 / 3 /

5

/ 2

CRABP2

/ 19 / 17 /

/ -
LEFTB / 18 / - /

/ -

CCNB1

/ 10 / 2 /

/ 2

CCNC

/ 9 / 3 /

/ 3

SFRP2

/ 8 / 3 /

/ 2

IMP-2

/ 8 / 3 /

/ 5

TK1

/ 7 / 3 /

/ 3

SET

/ 6 / 2 /

/ 3

BRIX

/ 6 / 3 / 4 / 3

CRABP1

/ 5 / 3 /

/ 11

STK12

/ 4 / 3 /

/ 2

MAD2L2

/ 4 / 2 /

/ 5

PTTG1

/ 6 / 3 /

/ 5

NME2

/ 11 / 7 /

/ 8

CDC2

/ 9 / 3 /

/ 7

Metabolism

DNA & RNA
NPM1 / 23 / 13 /

/ -
SSB / 20 / 6 /

/ 6

DDX21

/ 14 / - /

/ 4

/ 9 / 3 /

/ 4

Jade-1

/ 8 / 2 /

/ 2

RAMP

/ 6 / - /

/ -

HMGB2

/ 6 / 3 /

/ 4

EPRS

/ 5 / - / 5 / 2

RPL7

/ 4 / 3 / 4 / 4
SNRPF / 23 / 6 /

/ 6

EIF4A1

/ 18 / 9 /

/ 6

RPL6

/ 17 / 8 /

/ 8

HMGIY

/ 12 / 10 /

/ 15

HNRPAB

/ 12 / 5 /

/ 4

KPNA2

/ 11 / 5 /

/ 6

NBR2

/ 8 / 7 /

/ 6

RPL24

/ 6 / 3 /

/ 8

RPL4

/ 6 / 3 / 3 / 10

Metabolism

SLC16A1

/ 19 / 5 / 10 / 5
ELOVL6 / 19 / 2 / 12 / 2

CYP26A1

/ 18 / 2 / 14 / 2

FABP5

/ 18 / 4 / 4 / 2

KIF4A

/ 11 / 2 / 4 / -

PSMA2

/ 10 / 5 / 4 / 4

MGST1

/ 7 / 2 / 3 / 2

MTHFD2

/ 6 / - / 6 / 2

MTHFD1

/ 5 / 2 / 5 / 2

HDAC2

/ 5 / 3 / 5 / 4

IDH1

/ 5 / - / 4 / -
TDGF1 / 5 / - / 7 / 2

HSSG1

/ 5 / - / 3 / -

LAPTM4B

/ 3 / - / 4 / -

LDHB

/ 15 / 11 / 4 / 9

SMS

/ 14 / 4 / 4 / 5

PSMA3

/ 6 / 4 / 3 / 5

IMPDH2

/ 6 / 5 / 4 / 5

CCT8

/ 12 / - / 4 / 5

Novel genes

IFITM1

/ 12 / - / 5 / 2

GSH1

/ 12 / 3 / 5 / 2

PPAT

/ 9 / 2 / 6 / -

KIAA1573

/ 9 / 2 / 4 / -

C20orf1

/ 8 / - / 5 / 4

Laminin receptor

/ 8 / 6 / 4 / 3

C20orf129

/ 5 / - / 5 / 4

ARL8

/ 4 / - / 5 / -

Numatrin

/ 17 / 13 /

/ 16

C20orf168/ HNRPA1

/ 10 / 5 / 5 / 7

TD-60

/ 9 / 4 / 3 / 7

C15orf15

/ 8 / 5 / 4 / 7

Others

NASP

/ 10 / 2 / 7 / 2

LRRN1

/ 8 / 2 / 8 / 3

Other ES cell markers

CER1

/ 44 / - / 12 / -
DNMT3B / 22 / 2 / 34 / -
SOX2 / 4 / 3 / 5 / 3

a Numbers represents fold expression compared to HuURNA.

b (-) Indicates not expressed.

77/92 genes showed down modulation in Day 13 BG02 -EB and out of them 53 showed down modulation in PEB. Bold genes showed reverse pattern in PEB (over expression in PES) compared to day 13 BG02 EB.

Table-2: Genes that were upregulated in Day 13 EBand Pooled samples of EB

Gene / BG02-ES / Day 13 BG02-EB / Pooled ESa / Pooled EBa

KRT8

/ 17 / 45 /

KRT18 / 23 / 24 /

TUBB5

/ 5 / 8 / 6 / 11

ACTC

/ 22 / 23 / 4 / 6

RPLP0

/ 7 / 8 / 7 / 9
SERPINH1 / 6 / 8 / 4 / 10

TUBB4

/ 4 / 5 / 4 / 7

RPS24

/ 15 / 17 / 5 / 18

H-plk /Znf257

/ 6 / 7 / 5 / 4

PITX2

/ 7 / 11 / 6 / 4

Numbers indicate fold expression compared to HuURNA.

aPEB and PES were derived from GE01, GE07 and GE09 ES cell lines. Bold genes showed a reverse pattern of expression in PEB compared to day 13 EB

Table-3: Categorization of 194 genes expressed uniquely in Day 13 EB and pooled samples of EB at  3 folds but not in BG02-ES and pooled samples of ES

Categoryb

/ Genea
ES cell differentiation specific genes
(12) / ACIN1, DPPA4, COL5A2, COL1A2, COL4A2, COL6A2, COL6A3, P66ALPHA, PABPN1, S100A13, TMSB4X, TWSG1
Cellular process (Cell signaling, Cell growth, Cellular process, Cell cycle) (69) / ARID4B, ATOX1, ARF6, BST2, CALR, CCND2, CDKN1C, CNN2, CD164L1, CD99, CDC34, CDC42, CKLFS6, CNN3, COPZ1, DDX43, ERBB2, GNAS, Glypican-3, GPR48, G22P1, GPR43, GSPT1, HIVEP3, HYPC, IMP-4, ITM2C, IGFBP2, IL11, IL26, ILF2, ITPK1, JTB, JUP, KCNA5, KCTD10, KDELR1, LRP5, LRP6, LY6E, MCF2L, MYBL2, MFGE8, NAALD2, NID2, NME4, NEO1, NMI, PRKAG1, PRKWNK3, PSG9, PDCD4, RAB1A, RAB23, RAB27A, RRBP1, SEC61A1, SIAHBP1, SLC40A1, SLC9A3R1, TGFB1, TRIF, TIMM17B, TMEM30A, TRIM34, VCIP135, VAT1, VAPA, YWHAE
Cytoskeleton or cell motility (6) / ARPC1B, CFL1, GYPC, SPTA1, TUBA4, VIM
Metabolic activity
DNA and RNA related (15) / ATF5, GABARAP, GLE1L, GNB2L1, HNRPH3, , RPL17, RPL18, RPL28, RPL36, RPLP2, RPS11, RPS17, RPS29, RPS3, ZC3HAV1
Metabolism (55) / APG4B, ARHGAP9, APOA1, BG1, COX6B, COX7C, C5ORF15, C11ORF9, C13ORF12, C14ORF47, C14ORF58, C6orf157, COBLL1, CXX1, CHST2, DRG1, DSCR4, DXYS155E, EIF4EBP1, FAM36A, FBXL14, GBA, GOLGA6, H3F3B, H2AFY2, HSPC051, LEPREL2, MCM8, MMP2, MRC2, MIF, NME4, FADS1, PDHA1, PLEKHJ1, PMS2L9, POP1, PPIA. PRDX2, PRKDC, PRR3, PHGDH, PLTP, PPP1CA, RAB11F1P1, S100A11, STARD3, STK36, TTC3, UBA52, UBE2E3, UQCR, VNN3, VTN, XYLT2
Others (37) / ZNF306, ZNF350
26 Hypothetical proteins and 9 unknown proteins

a indicates unique genes expressed at a 99% confidence interval ( 3 fold) in EBs. b numbers in parenthesis represent the number of genes under each category.
Table-4: Categorization of 37 unknown genes expressed uniquely in EBa

Gene / Blast symbol / Chrom. Location / Unigene /
Accession
No. / Predicted protein size / Annotation
DKFZP761A052 / Xp11.23 / Hs.184029 / 571aa / Hypothetical protein DKFZp761A052
FLJ10134/
DERP7 / 3q12.3 / Hs.104800 / 275aa / Hypothetical protein FLJ10134 / dermal papilla derived protein 7
FLJ12541/
Stra6 / 15q22.33 / Hs.24553 / 667aa / FLJ12541 / stimulated by retinoic acid gene 6
FLJ12650 / 1p35.1 / Hs.436090 / 163aa / Hypothetical protein FLJ1260
FLJ12983 / 7 / AK023045 / 177aa / Hypothetical protein FLJ12983 / LOC340351
FLJ14129 / 8q24.3 / Hs.512740 / 374aa / Hypothetical protein FLJ14129
FLJ21031 / 1p31.3 / AK024684 / 126aa / Hypothetical protein FLJ21031 : Homo sapiens cDNA:clone CAE07336
FLJ22329 / 19p13.12 / Hs.418795 / 622aa / Hypothetical protein FLJ22329
FLJ22761 / 10q22.1 / Hs.445459 / 911aa / Hypothetical protein FLJ22761
FLJ23233 / 19q13.43 / Hs.98593 / 387aa / Hypothetical protein FLJ23233
FLJ14213 / 11p13 / Hs.75307 / 213aa / Hypothetical protein FLJ14213
H1FX / 3q21.3 / Hs.75307 / 213aa / H1 histone family , member X may be related to metabolism
KIAA0146 / 2q13 / Hs.381058 / 45aa / KIAA0146 protein (KIAA0146), mRNA
KIAA1052 / 11q23.3 / Hs.18624 / 1460aa / KIAA1052 protein
KIAA1318/
RGAG1 / Xq23 / Hs.20171 / 1388aa / KIAA1318/ Retrotransposon gag domain containing 1 (RGAG1)
KIAA1602 / 2q13 / Hs.143067 / 1189aa / KIAA1602
LOC347544 / Xq28 / XP_293412 / 147aa / Predicted: Homo sapiens similar to ribosomal protein L18a; 60S ribosomal protein L18a (LOC347544)
LOC348262 / 17p13.3 / Hs.285165 / 175aa / Hypothetical proteins LOC348262
LOC51215/
PRO0398 / 19 / AF113674.1 / 105aa / Clone FLB1727 / C3: complement component 3
LOC55971 / 7q22.1 / Hs.285165 / 500aa / Insulin receptor tyrosine kinase substrate (LOC55971), mRNA
MDS025/
MDS011 / 11q14.1 / Hs.368866 / 254aa / Hypothetical protein MDS025/ Hypothetical protein MDS011
MGC35097 / 3p21.31 / Hs.13781 / 354aa / Hypothetical protein MGC35097
MGC51082 / 19q13.32 / Hs.99093 / 188aa / Hypothetical protein MGC51082
MGC8721/ XTP-3 / 8p12 / Hs.27921 / 339aa / Hypothetical protein MGC8721/ XTP-3 / FLJ22274
PF20 / 2q34 / Hs.6783 / 631aa / PF 20 / WD repeat domain 29 (sperm associated WD repeat proteins)
POF1B / Xq21.2 / Hs.267038 / 589aa / Premature ovarian failure 1B (POF1B)
PRO1776 / 5 / -b / - / DISCONTINUED in mAdb : Locus ID 55377 was defined by AF119853.1
PRO2792 / 11q23.3 / AF180681 / 1544aa / Hypothetical protein PRO2792 / ARHGEF12 : Rho guanine nucleotide exchange factor (GEF) 12
PRO2852 / 9 / Hs.493756 / 502aa / UBAP1 : Ubiquitin associated protein 1
RANBP10/ RANBP9 / 16q22.1 / Hs.6343 / 620aa / Ran-binding protein 10 / RANBP9 / RANBPM : encodes a protein that binds RAN, a small GTP binding protein belonging to the RAS superfamily that is essential for the translocation of RNA
RSHL2 / RSP3 / 6q25.3 / Hs.154628 / 560aa / Radial spokehead-like 2 (RSHL2) : Radial spoke protein 3 or A-kinase anchoring protein (AKAP) involved in neuronal migration during development of CNS
UNC13D / UNC13 / 17q25.3 / Hs.41045 / 1090aa / Unc-13 homolog D (C.elegans) may be involved in synaptic transmission
ZFHX1B / 2q22 / Hs.34871 / 1214aa / Zinc finger homeobox 1b encodes for SMAD interacting protein 1: considered an important gene for normal embryonic neural crest development. Mutation in this gene causes Hirschsprung's disease.
ZC3HAV1 / 7q34 / Hs.133512 / - / zinc finger CCCH type, antiviral 1(ZC3HAV1),transcript variant 2, mRNA.
ZNF306 / 6p21.33 / Hs.66774 / 538aa / Zinc finger protein 306 : involved in transcriptional regulation
ZNF350 / 19q13.41 / Hs.407694 / 532aa / Zinc finger protein 350 : involved in transcriptional regulation
ZP2 / 16p12 / Hs.73982 / 745aa / Zona pellucida glycoptorein 2 (sperm receptor) : may be involved with fertilization and preimplantation development

aTwenty six hypothetical proteins, 2 zinc finger proteins and 9 unknown genes over expressed in EB were analyzed by using bioinformatic tools available at multiple databases, including NCBI (National Center for Biotechnology) blast search, locus link, pubmed, the National Cancer Institute and the Center for Information Technology, National Institute of Health. Gene symbol or blast search name, human unigene or gene bank accession number, chromosomal localization, predicted protein size and possible functions are shown. aa indicates amino acids. b(–) indicates not found.
Table-5: Comparison of expression of some known ES marker by microarray and MPSS

Gene / MPSS / Microarray / Microarray

Undifferentiated cell markers

PES/PEBb

PES/PEBa

BG02/Day 13/Day 21a

DNMT3B / 1274/56 / 34/- / 22/2/5
POU5F1 / 658/20 / 23/3 / 28/3/4
GJA1 / 452/406 / 6/2 / 10/4/3
Galanin / 221/28 / 5/4 / 33/4/3
NANOG / 16/- / 8/- / 6/0/9
LEFTB / 72/- / 24/- / 18/-/-
TDGF1 / 38/- / 7/2 / 5/0/2

a Indicates fold expression by microarray analysis compared to HuURNA.

b Indicates transcripts per million (tpm) by MPSS analysis.
Table-6: Genes showed overexpression by microarray and EST

Enumeration in pooled samples of EB compared to pooled samples of ES

Gene name / Accession number / EST-enumeration PES/PEBa / Microarray
PES/ PEBb
EB specific markers
KRT8 / X74929 / 9/71 / 4/27
KRT18 / M26326 / 8/46 / 5/23
TUBB-5 / AK001295 / 5/8 / 6/11
ACTC / J00073 / 1/4 / 4/6
TUBB4 / AK001295 / ND / 4/7
Cell cycle regulation
IMP-2 / NM_006548 / 9/14 / 5/7

NME2

/ M36981 / 3/6 / 3/8
Metabolism DNA RNA related
RPLP0 / AK001313 / 154/167 / 7/9
EIF4A1 / D13748 / 31/36 / 4/6

RPL24

/ M94314 / NDc / 4/6

RPL4

/ D23660 / ND / 3/10

SNRPF

/ X85372 / ND / 3/6

HMGIY

/ L17131 / ND / 9/15
Metabolic activity
SERPINH1 / D83174 / 28/32 / 4/10
CCT8 / D13627 / 8/10 / 4/5
SMS / AD001528 / 5/7 / 4/5

Novel genes

C15orf15

/ NM_016304 / 2/6 / 4/7

TD-60

/ AB040903 / ND / 3/7

C20orf168 / HNRPA1

/ AL050348 / ND / 5/7

Numatrin

/ AL353580 / ND / 4/7

a Ratio of fold expression in pooled samples of ES and EB by EST enumeration

b Indicates gene expression ratio of Pooled samples of ES and EB by microarray.

c ND, expression not detected.

Table-7: Genes overexpressed by MPSS, microarray and EST enumeration in

Day 13 EB compared to BG02-ES

Gene name / Accession number / EST-enumeration PES/PEBa / Microarray BG02-ES / Day 13EBb / MPSS
BG02-ES/ Day 13EBc
EB specific markers
KRT8 / X74929 / 9/71 / 17/45 / 594/2472
KRT18 / M26326 / 8/46 / 23/24 / 323/2090
TUBB-5 / AK001295 / 5/8 / 5/8 / ND
ACTC / J00073 / 1/4 / 22/23 / 112/141
TUBB4 / AK001295 / NEd / 4/5 / NDe
Cell cycle regulation
PITX2 / AF048722 / NE / 7/11 / ND
Metabolism DNA RNA related
RPLP0 / AK001313 / 154/167 / 7/8 / ND
Metabolic activity
SERPINH1 / D83174 / 28/32 / 6/8 / ND

a Indicates EST enumeration ratio of pooled samples of ES and EB.

b Indicates gene expression ratio of Pooled samples of ES and EB by microarray

c Indicates tpm level by MPSS analysis in BG02 ES and day 13 EB.

d NE indicates no expression observed by EST enumeration.

e ND indicates not done.

Table-8:Comparison of expression profile of 8 unknown genes by MPSS and microarray

Gene / Description / MPSS
ES /EB (tpm)a / Microarray PES/PEB b / Microarray
BG02-ES/D-13 EB/ D21-EB b
FLJ10134 / Hypothetical protein FLJ10134 / 12/145 / -/3 / -/3/2
FLJ12541 / Hypothetical protein FLJ12541 / 0/26 / -/3 / -/3/-
FLJ22329 / Hypothetical protein FLJ22329 / 5/86 / -/4 / 2/3/2
LOC348262 / Hypothetical proteins LOC348262 / 51/103 / -/3 / -/3/2
MDS025 / Hypothetical protein MDS025 / 9/15 / 2/5 / 2/3/2
MGC35097 / Hypothetical protein MGC35097 / 0/17 / -/4 / -/3/2
MGC51082 / Hypothetical protein MGC51082 / 18/40 / -/3 / -/3/2
MGC8721 / Hypothetical protein MGC8721/ XTP-3 / FLJ22274 / 83/101 / -/3 / -/3/-

aindicates the expression of these genes in pooled ES and EB by MPSS analysis at tpm level. b indicates the fold expression of these genes in both ES and EBs by microarray.

(-) indicates ≤ 1 fold or no expression.

Table-9: Categorization of 46 genes expressed uniquely in Day 13 EB and pooled samples of EB by microarray but not by MPSSa

Categoryb

/ Gene
ES cell differentiation specific genes (1) /

TWSG1

Cellular process (Cell signaling, Cell growth, Cellular process, Cell cycle) (11) / DDX43, ERBB2, GPR43, HIVEP3, IL26, KCNA5, MYBL2, NID2, RAB27A, SLC9A3R1, TRIM34
Cytoskeleton or cell motility (2) / SPTA1, TUBA4
Metabolic activity
DNA and RNA related (2) / KIAA1318, RPL18
Metabolism (21) / ARHGAP9, C5ORF15, C11ORF9, C14ORF147, CHST2, DSCR4, DXYS155E, GBA, GOLGA6, LEPREL2, LOC347544, LOC51215, MCM8, PRKWNK3, PRO1776, PRO2792, PRO2852, S100A11, UBE2E3, VNN3, VTN
Unknown genes (9) / ZNF306, ZNF350, FLJ12650, FLJ12983, FLJ21031, FLJ23233
H1FX, POF1B, ZP2

aGenes expressed at 99% confidence interval ( 3 fold) are shown. Expression of the genes in italics is confirmed by Agilent human 22k oligo-array (G4110B). bNumbers in parenthesis represent the number of genes under each category.

Table-10: Gene expression in Day 13 EBand Day 21 EBa

Gene / BG02-ES / Day 13 BG02-EB / Day 21 BG02-EB

KRT8

/ 17 / 45 /

KRT18 / 23 / 24 /

TUBB5

/ 5 / 8 / 2

ACTC

/ 22 / 23 / 1

RPLP0

/ 7 / 8 / 6
SERPINH1 / 6 / 8 / 1

TUBB4

/ 4 / 5 / 2

RPS24

/ 15 / 17 / 17

a Indicates fold expression compared to HuURNA.